It is increasingly desirable to provide a rapid high sensitivity system to detect low levels of ligands in body fluids, plant extracts, environmental samples, tissue samples and enrichment broths. Ideally, such systems should have a minimal number of procedural steps and yield reliable results, even when used by untrained persons.
To a significant extent, many known tests presently available for detecting ligands are either time consuming, labor intensive, or in need of instrumental assistance to read results. Most known tests also lack an acceptable degree of sensitivity or specificity. This is unfortunate since rapid testing is important for diagnosis and treatment of various bacterial, fungal, and viral pathogens. Additionally, rapid testing can be used for detection of drug analytes, cancer cells, antibodies, disease-state protein, and the like.
Although known types of ligand-receptor assays have been used to detect the presence of various substances, such as ligands, there is a need in the art to provide a rapid, high sensitivity assay requiring a minimum degree of skill from a user. Rapid test assay devices for field use, such as in a home or doctor's office are known in the art for detecting proteins, peptides, drugs, carbohydrates, haptens, chemicals, chemical reaction with intermediate compounds, and the like. Such devices are referred to as one-step lateral flow or immuno-chromatographic assays. These types of assay devices require a minimal number of steps and can be performed by an untrained person.
Several one-step lateral flow immunoassay devices having a strip capable of transporting a developing liquid by capillary action having a first zone for receiving a sample, a second zone impregnated with a first reagent capable of being transported by the developing liquid, and a third zone impregnated with a second reagent, are known in the art.
U.S. Pat. Nos. 4,094,647 and 4,235,601 (Deutsch et al.) disclose a test device for determining a characteristic of a sample, particularly for determining substances in fluid samples. A strip element has a predetermined location for receiving the test sample and predetermined locations incorporated with reagent means for providing a detectable response sensitive to the characteristic under determination. The beginning end portion of the strip element is immersed in the developing fluid which, as a result, traverses the length of the strip element, thereby promoting appropriate contact between the test sample and the reagent means resulting in the disposition of a detectable response at a predetermined location on the strip element. The test device is particularly suited for performing binding assays, and in particular those wherein a radioisotope is used as a label, such as radioimmunoassays.
U.S. Pat. No. 6,352,862 (Davis et al) discloses an analytical test device incorporating a dry porous carrier to which a liquid sample can be directly applied. The device also incorporates a labeled specific binding reagent which is freely mobile in the porous carrier when in the moist state, and an unlabelled specific binding reagent which is permanently immobilized in a detection zone on the carrier material. The labeled and unlabelled specific binding reagents are capable of participating in either a sandwich reaction or a competition reaction in the presence of the analyte, in which prior to the application to the device of a liquid sample suspected of containing the analyte, the labeled specific binding reagent is retained in the dry state in a macroporous body, eg. of plastics material having a pore size of 10 microns or greater, through which the applied liquid sample must pass en route to the porous carrier material, with the labeled specific binding reagent being freely soluble or dispersible in any liquid sample which enters the macroporous body.
U.S. Pat. No. 5,120,643 (Ching et al.) relates to improved specific binding assay methods, kits and devices utilizing chromatographically mobile specific binding reagents labelled with colloidal particles. Specific binding reagents labelled with colloidal particles such as gold and selenium may be subjected to rapid chromatographic solvent transport on chromatographic media by means of selected solvents and chromatographic transport facilitating agents. Further, impregnation of solid substrate materials with labile protein materials including colloidal particle and enzyme labelled reagents in the presence of meta-soluble proteins provides for the rapid resolubilization of such materials which have been dried onto such substrate materials.
A major disadvantage of all of the known prior art is that the majority of sample applied to a lateral flow assay is not involved in the immunological reaction. For example, between 10 .mu.l to 20 .mu.l of sample is all that is required to fully rehydrate a 5 by 5 mm gold pad or membrane with the conjugate pad or membrane composed of polyester, glass, nylon, cellulose or other fibers. Since the conjugate pad or membrane is in the device flow path and in contact with the membrane, a minimal remaining sample is involved in the immunological reaction. The remaining 50 .mu.l to 150 .mu.l of sample acts as a liquid front by pushing the reacted analyte conjugate complexes through the flow path. Since only 10 to 20 microliters of sample is involved in the reaction, only a few analyte molecules of interest in the sample are bound to the colloidal conjugate, thus limiting the number of molecules involved in the immuno-chromatographic reaction. Thus, a severe limitation of the one-step lateral flow assay is the limited amount of sample actually involved in the reaction with the conjugate.
Further, in a one-step lateral flow assay, as a sample mixes with a dry conjugate, incomplete mixing often yields conjugate particles with vastly different numbers of captured analyte molecules. This alone may be a source of decreased sensitivity since the maximum number of analyte molecules are not captured on the surface of the conjugate particles.
Other designs are known in the art also include two-step assays. These assays often require the conjugate pad or membrane to be physically and spatially removed from the test strip. For example, there are assays known in the art where the conjugate is contained in book form, or a “male to female” molded apparatus. In the book form, the test strip is located on one panel of the book while the conjugate is located on the other panel. The test requires the user to close both halves for the test to begin. With the “male to female” apparatus, the sample is applied to the male portion of the apparatus, and the two halves are closed to initiate the reaction. Another format has the conjugate dried and located on a separate sampling stick. The sampling stick is mixed with a sample and is then fixed in the cassette sample well. For example, some two step assay platforms are known in the art, such as indicated in U.S. Pat. No. 6,824,997 and U.S. Pat. No. 5,418,171.
These tests have a significant drawback though. The end user must perform an additional step to physically bring the membrane and conjugate into contact after the sample has been adequately mixed. Sometimes, the sample “swamps” the assay, making the results hard to read.
Thus, there is a desire and need in the art for a high sensitivity assay system and apparatus to rapidly detect low levels of ligands in a small sample size of fluid. Such new tests should involve a minimal number of procedural steps while at the same time, yielding reliable results, even when used by untrained persons.